Abstract Opioid and catecholamine receptors are key regulators of neurophysiology and behavior, and are important targets of therapeutic and abused drugs. These receptors are fundamentally regulated by endocytosis and specific membrane trafficking events in the endocytic pathway. Endocytic membrane trafficking has long been recognized to influence adaptation or maladaptation of the endogenous opioid and catecholamine systems to chronic or repeated drug administration. It is now evident that endocytic trafficking of receptors also impacts the acute response and may thereby differentiate the actions of therapeutically and addiction - relevant drugs over a wide time frame. In the previous funding period we defined much of the biochemical machinery determining the endocytic regulation of opioid receptors, and established effects on both long-term and acute signaling. The proposed studies build on progress made in the previous funding period to elucidate a fundamentally new mechanism of opioid and adrenergic receptor signaling based on receptor activation in internal membrane compartments as well as the plasma membrane. We propose to (1) Define mechanisms that limit GPCR signal strength or duration from endosomes; (2) Determine how endocytosis facilitates downstream transcriptional control by GPCRs; and (3) Delineate membrane trafficking properties of opioid receptors in axons.